CportucalensisElectrocompetentCells

Prepare 100 mL sterile LB in a 500 mL Erlenmeyer flask.

Prepare ice-cold 10 % glycerol and ultrapure water (both sterile).

Two days prior to electroporation, streak out strain on LB agar and grow at 30 ºC.

One day prior to electroporation, inoculate a 5 mL LB liquid culture with a patch of cells from the overnight streak and incubate slanted, shaking at 250 rpm at 30 ºC overnight.

Day of the electroporation, inoculate 1 mL of the overnight culture into the 100 mL LB flask and grow to OD600 = 0.4, shaking at 250 rpm at 30 ºC.

Chill flask on ice for 20 minutes.

Wash two the culture two times into ice-cold water (5000 x g for 10 minutes at 4 ºC; I typically divide the 100 mL volume into four 50 mL conical tubes, washing with 25 mL volumes).

Combine pellets and spin final time.

Resuspend in 2 mL ice-cold 10% glycerol.

Aliquot 50 µL volumes into ice-cold microcentrifuge tubes and flash-freeze in liquid nitrogen.

Store at -80 ºC or use immediately.

Two days prior, streak out strain as above.

One day prior, grow 5 mL LB culture overnight as above.

Day of, inoculate 50 µL overnight culture into 5 mL LB and incubate slanted, shaking at 250 rpm at 30 ºC until OD600 = 0.3.

Chill on ice for 10 minutes.

Wash three times into ice-cold water, final resuspension of combined pellets 50 µL (can also spin full 5 mL culture in conical tube directly).